Regulation of the proprotein convertase subtilisin/kexin type 9 in intestinal epithelial cells

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally promotes the degradation of the low-density lipoprotein receptor (LDLr) in hepatocytes and increases plasma LDL cholesterol. It is not clear, however, whether PCSK9 plays a role in the small intestine. Here, we characterized the patterns of variations of PCSK9 and LDLr in fully differentiated Caco-2/15 cells as a function of various potential effectors. Cholesterol (100 microM) solubilized in albumin or micelles significantly downregulated PCSK9 gene (30%, P<0.05) and protein expression (50%, P<0.05), surprisingly in concert with a decrease in LDLr protein levels (45%, P<0.05). Cells treated with 25-hydroxycholesterol (50 microM) also displayed significant reduction in PCSK9 gene (37%, P<0.01) and protein (75% P<0.001) expression, whereas LDLr showed a decrease at the gene (30%, P<0.05) and protein (57%, P<0.01) levels, respectively. The amounts of PCSK9 mRNA and protein in Caco-2/15 cells were associated to the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein-2 (SREBP-2) that can transcriptionally activate PCSK9 via sterol-regulatory elements located in its proximal promoter region. On the other hand, depletion of cholesterol content by hydroxypropyl-beta-cyclodextrin upregulated PCSK9 transcripts (20%, P<0.05) and protein mass (540%, P<0.001), in parallel with SREBP-2 protein levels. The addition of bile acids (BA) taurocholate and deoxycholate to the apical culture medium lowered PCSK9 gene expression (25%, P<0.01) and raised PCSK9 protein expression (30%, P<0.01), respectively, probably via the modulation of farnesoid X receptor. Furthermore, unconjugated and conjugated BA exhibited different effects on PCSK9 and LDLr. Altogether, these data indicate that intestinal PCSK9 is highly modulated by sterols and emphasize the distinct effects of BA species.

CFTR knockdown stimulates lipid synthesis and transport in intestinal Caco-2/15 cells

“…RNA was isolated from differentiated Caco-2/15 cells using Trizol (Invitrogen, Carlsbad, CA) according to manufacturer's instructions as described previously (38,50,63). A quantity of 2 g of total RNA was reverse transcribed by using the Moloney murine leukemia virus reverse transcriptase (Invitrogen).…”

“…Total lysate proteins (20 g) were separated on SDS-PAGE and electroblotted onto nitrocellulose membranes as described previously (38,60). After 1 h of blocking in 5% nonfat dry milk at room temperature, membranes were incubated with one of the following primary antibodies: 1:1,000 anti-CFTR (Cellular Signaling, Danvers, MA), 1:15,000 anti-SRB1, 1:5,000 anti-NPC1L1, 1:500 anti-ABCG8 (all from Novus, Littleton, CO), or 1:40,000 mouse monoclonal anti-␤ actin antibody (Sigma) overnight at 4°C.…”

Pcsk9

“…12 PCSK9 regulation of the LDLR protein levels in intestinal cell lines has been reported. 69,115 The possible implication of PCSK9 in the transintestinal reverse cholesterol excretion from the small intestine has been recently evoked. 116 Although the LDLR is involved in this process, it seems that yet another receptor(s) sensitive to PCSK9 is/are also implicated.…”

New Insights In Intestinal Sar1B GTPase Regulation and Role in Cholesterol Homeostasis

“…Forcing Sar1B resulted in the concomitant down‐regulation of PCSK9 and LDLR. These results could appear as a paradox but this phenomenon was previously described by our group: we could demonstrate that PCSK9 and LDLR are either stimulated or down‐regulated simultaneously via the SREBP‐2 transcription factor [Leblond et al, ]. Also, Engelking et al recently showed that, in mice and in contrast to liver, the increased jejunal PCSK9 expression did not define the increase in LDLR protein in response to ezetimibe and that IDOL, an E3 ubiquitin ligase that promotes LDLR degradation, trumped PCSK9 in the regulation of jejunal LDLR [Engelking et al, ].…”